EXCEED THE SPACE PROVIDED. Extracellular modulation of growth factor signaling represents a central mechanism for regulating the distribution and levels of ligand activity in development. In work supported by the previous funding period of this competitive renewal, we have shown that the EGF-CFC gene Cripto is required for anterior-posterior (A-P) axis patterning and embryonic mesoderm formation at early stages of mouse development. Our studies have provided genetic evidence for a model in which Cripto acts as an essential co-receptor for signaling by Nodal, a member of the transforming growth factor-beta (TGF-P) family that has multiple roles in formation of the vertebrate body plan. In the current application, we propose to investigate the functions of Cripto in A-Paxis patterning, mesoderm formation, and axial midline development, as well as its potential role in cardiogenesis. Based on our preliminarydata, we anticipate that these functions of Cripto involve Nodal-dependent activities as well as Nodal-independent activities that may reflect its cooperation with other specific members of the TGF- P family. In addition, our in vivo analyses will be complemented by cell culture and biochemical investigations of the mechanism of EGF-CFC cooperation with TGF-P signaling. We will pursue the following specific aims: I) Analysis of Cripto function in A-P axis patterning and mesoderm specification through phenotypic and chimera analysis of Cripto function at pre-gastrulation and gastrulation stages, and by microarray analysis to identify genes downstream of Cripto function. II) Investigation of the role of Cripto in axial midline formation by analysis of Cripto reduction-of-function mutants in the mouse and gain-of-function approaches in the chick embryo. Ill) Examination of the role of Cripto in cardiac development by tissue-specific targeted deletion of Cripto in cardiac progenitors in vivo, and investigation of the differentiation of Cripto- deficient EScells in culture. IV) Analysis of EGF-CFC activities in a cell culture assay system to investigate the Nodal-dependent and Nodal-independent activities of Cripto, and to identify TGF-P factors that can interact with EGF-CFC proteins. Taken together, our continuing studies of Cripto should provide important insights into the molecular mechanisms of extracellular modulation of morphogenetic signals.